S-nitrosylated human serum albumin-mediated cytoprotective activity is enhanced by fatty acid binding.
Binding of oleate to S-nitrosylated human serum albumin (SNO-HSA) enhances its cytoprotective effect on liver cells in a rat ischemia/reperfusion model. It enhances the antiapoptotic effect of SNO-HSA on HepG2 cells exposed to anti-Fas antibody. To identify some of the reasons for the increased cytoprotective effects, additional experiments were performed with glutathione and HepG2 cells. As indicated by 5,5'-dithiobis-2-nitrobenzoic acid binding, the addition of oleate increased the accessibility of the single thiol group of albumin. Binding of increasing amounts of oleate resulted in increasing and more rapid S-transnitrosation of glutathione. Likewise, binding of oleate, or of a mixture of endogenous fatty acids, improved S-denitrosation of SNO-HSA by HepG2 cells. Oleate also enhanced S-transnitrosation by HepG2 cells, as detected by intracellular fluorescence of diaminofluorescein-FM. All of the S-transnitrosation caused by oleate binding was blocked by filipin III. Oleate also increased, in a dose-dependent manner, the binding of SNO-HSA labeled with fluorescein isothiocyanate to the surface of the hepatocytes. A model in two parts was worked out for S-transnitrosation, which does not involve low molecular weight thiols. Fatty acid binding facilitates S-denitrosation of SNO-HSA, increases its binding to HepG2 cells and greatly increases S-transnitrosation by hepatocytes in a way that is sensitive to filipin III. A small nitric oxide transfer takes place in a slow system, which is unaffected by fatty acid binding to SNO-HSA and not influenced by filipin III. Thus, fatty acids could be a novel type of mediator for S-transnitrosation.
Ishima
Yu
Y
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan.
Akaike
Takaaki
T
Kragh-Hansen
Ulrich
U
Hiroyama
Shuichi
S
Sawa
Tomohiro
T
Suenaga
Ayaka
A
Maruyama
Toru
T
Kai
Toshiya
T
Otagiri
Masaki
M
D000818:Animals; D045744:Cell Line, Tumor; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005227:Fatty Acids / Q000737:chemistry*; D005372:Filipin / Q000737:chemistry; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D008297:Male; D009569:Nitric Oxide / Q000737:chemistry; D009584:Nitrogen / Q000737:chemistry*; D019301:Oleic Acid / Q000737:chemistry; D051381:Rats; D015427:Reperfusion Injury; D012709:Serum Albumin / Q000737:chemistry*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D005227:Fatty Acids; D012709:Serum Albumin; D013439:Sulfhydryl Reagents; D019301:Oleic Acid; D009569:Nitric Oxide; D005372:Filipin; D004228:Dithionitrobenzoic Acid; D009584:Nitrogen